Bentgrass event ASR-368 and compositions and methods for detection thereof

ABSTRACT

The present invention provides a bentgrass ASR-368 plant and seed. Also provided are assays for detecting the presence of the bentgrass ASR-368 based on a DNA sequence and the use of this DNA sequence as a molecular marker in a DNA detection method.

This application is a §371 U.S. national phase application ofInternational Application No. PCT/US2003/0038268 filed Dec. 3, 2003, andclaims the benefit of priority to U.S. Provisional Application No.60/431,153, filed Dec. 5, 2002.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology.More specifically, the invention relates to a glyphosate tolerantbentgrass plant event ASR-368 and to assays and methods for detectingthe presence of bentgrass plant event ASR-368 DNA in a plant sample andcompositions thereof.

BACKGROUND OF THE INVENTION

Bentgrass (Agrostis stolonifera) is an important turf species in manyareas of the world. The methods of biotechnology have been applied tobentgrass for improvement of the agronomic traits. One such agronomictrait is herbicide tolerance, in particular, tolerance to glyphosateherbicide. The control of weeds in bentgrass is particularlyproblematic. Bentgrass used on golf greens is especially sensitive tomany herbicides that are normally used on other turfgrasses or on otherareas of a golf course. Annual grasses, such as, crabgrass, foxtail,dallisgrass, and goosegrass must be controlled by use of a variety ofherbicides including bensulide, dithiopyr, oxadiazon, fenoxaprop andprodiamine applied at specific rates, environmental conditions, andseasons by expert applicators in order to be effective. Annual andperennial broadleaf weeds may be controlled in bentgrass turf byapplications of herbicides that include 2,4-D, MCPP, dicamba, andmixtures of these. Many grass and broadleaf herbicides cannot be used onbentgrass golf greens because of injury to the bentgrass, or they arenot registered for use on bentgrass. There is a need for a glyphosatetolerant bentgrass to replace the use of these herbicides and to providea method for effective grass and broadleaf weed control in bentgrassturf when glyphosate herbicide is applied.

N-phosphonomethylglycine, also known as glyphosate, is a well-knownherbicide that has activity on a broad spectrum of plant species.Glyphosate is the active ingredient of Roundup® (Monsanto Co.), a safeherbicide having a desirably short half-life in the environment. Whenapplied to a plant surface, glyphosate moves systemically through theplant. Glyphosate is phytotoxic due to its inhibition of the shikimicacid pathway, which provides a precursor for the synthesis of aromaticamino acids. Glyphosate inhibits the enzyme5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) found in plants.Glyphosate tolerance can also be achieved by the expression of bacterialEPSPS variants and plant EPSPS variants that have lower affinity forglyphosate and therefore retain their catalytic activity in the presenceof glyphosate (U.S. Pat. Nos. 5,633,435; 5,094,945, 4,535,060, and6,040,497).

The expression of foreign genes in plants is known to be influenced bytheir chromosomal position, perhaps due to chromatin structure (e.g.,heterochromatin) or the proximity of transcriptional regulation elements(e.g., enhancers) close to the integration site (Weising et al., Ann.Rev. Genet 22:421-477, 1988). For this reason, it is often necessary toscreen a large number of events in order to identify an eventcharacterized by optimal expression of a introduced gene of interest.For example, it has been observed in plants and in other organisms thatthere may be a wide variation in levels of expression of an introducedtransgene among events. There may also be differences in spatial ortemporal patterns of expression, for example, differences in therelative expression of a transgene in various plant tissues, that maynot correspond to the patterns expected from transcriptional regulatoryelements present in the introduced gene construct. For this reason, itis common to produce hundreds to thousands of different events andscreen those events for a single event that has desired transgeneexpression levels and patterns for commercial purposes. An event thathas desired levels or patterns of transgene expression is useful forintrogressing the transgene into other genetic backgrounds by sexualcrossing using conventional breeding methods. Progeny of such crossesmaintain the transgene expression characteristics of the originaltransformant. This strategy is used to ensure reliable gene expressionin a number of varieties that are well adapted to local growingconditions and market demands.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the premarket approval and labeling of foods derived fromrecombinant crop plants, for example. It is possible to detect thepresence of a transgene by any well known nucleic acid detection methodsuch as the polymerase chain reaction (PCR) or DNA hybridization usingnucleic acid probes. These detection methods generally focus onfrequently used genetic elements, such as promoters, terminators, markergenes, etc. As a result, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct unless the sequence of chromosomal DNAadjacent to the inserted DNA (“flanking DNA”) is known. Anevent-specific PCR assay is discussed, for example, by Windels et al.(Med. Fac. Landbouww, Univ. Gent 64/5b:459-462, 1999), who identifiedglyphosate tolerant soybean event 40-3-2 by PCR using a primer setspanning the junction between the insert and flanking DNA, specificallyone primer that included sequence from the insert and a second primerthat included sequence from flanking DNA. Event-specific DNA detectionmethods for a glyphosate tolerant corn event have also been described(US 20020013960 A1, herein incorporated by reference in it's entirety).

The present invention relates to a glyphosate herbicide tolerantbentgrass plant ASR-368 and to DNA compositions that comprise atransgene/genomic junction region contained in the genome of ASR-368 andto a method for detection of the transgene/genomic junction region inbentgrass plant ASR-368 and progeny thereof.

SUMMARY OF THE INVENTION

The present invention is a bentgrass transgenic event designated ASR-368having seed deposited with American Type Culture Collection (ATCC) withAccession No.PTA-4816. Another aspect of the invention is the progenyplants, or seeds, or regenerable parts of the plants and seeds of thebentgrass plant ASR-368. The invention also includes plant parts ofbentgrass plant ASR-368 that include, but are not limited to pollen,ovule, flowers, shoots, roots, and leaves.

One aspect of the invention provides compositions and methods fordetecting the presence of a transgene/genomic junction region frombentgrass plant event ASR-368. DNA molecules are provided that compriseat least one transgene/genomic junction DNA molecule selected from thegroup consisting of SEQ ID NO:1 and SEQ ID NO:2, and complementsthereof, wherein the junction molecule spans the insertion site thatcomprises a heterologous DNA inserted into the bentgrass genome and thegenomic DNA from the bentgrass cell flanking the insertion site inbentgrass event ASR-368. A bentgrass plant ASR-368 and seed comprisingthese molecules is an aspect of this invention.

A novel DNA molecule is provided that is a transgene/genomic region SEQID NO:3 or the complement thereof, wherein this DNA molecule is novel inbentgrass event ASR-368. A bentgrass plant and seed comprising SEQ IDNO:3 in the genome is an aspect of this invention.

According to another aspect of the invention, a DNA molecule is providedthat is a transgene/genomic region SEQ ID NO:4, or the complementthereof, wherein this DNA molecule is novel in bentgrass event ASR-368.A bentgrass plant and seed comprising SEQ ID NO:4 in the genome is anaspect of this invention.

According to another aspect of the invention, two DNA molecules areprovided for use in a DNA detection method, wherein the first DNAmolecule comprises at least 11 or more contiguous polynucleotides of anyportion of the transgene region of the DNA molecule of SEQ ID NO:3 and aDNA molecule of similar length of any portion of a 5′ flanking bentgrassgenomic DNA region of SEQ ID NO:3, wherein these DNA molecules when usedtogether are useful as DNA primers in a DNA amplification method thatproduces an amplicon. The amplicon produced using these DNA primers inthe DNA amplification method is diagnostic for bentgrass event ASR-368.Any amplicon comprising SEQ ID NO:1 produced by DNA primers homologousor complementary to any portion of SEQ ID NO:3 is an aspect of theinvention.

According to another aspect of the invention, two DNA molecules areprovided for use in a DNA detection method, wherein the first DNAmolecule comprises at least 11 or more contiguous polynucleotides of anyportion of the transgene region of the DNA molecule of SEQ ID NO:4 and aDNA molecule of similar length of any portion of a 3′ flanking bentgrassgenomic DNA of SEQ ID NO:4, where these DNA molecules are useful as DNAprimers in DNA amplification method. The amplicon produced using theseDNA primers in the DNA amplification method is diagnostic for bentgrassevent ASR-368. Any amplicon comprising SEQ ID NO:2 produced by DNAprimers homologous or complementary to any portion of SEQ ID NO:4 is anaspect of the invention.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding specifically to the bentgrass eventASR-368 DNA in a sample are provided. Such methods comprise: (a)contacting the sample comprising DNA with a primer set that, when usedin a nucleic acid amplification reaction with genomic DNA from bentgrassevent ASR-368 produces an amplicon that is diagnostic for bentgrassevent ASR-368 (b) performing a nucleic acid amplification reaction,thereby producing the amplicon; and (c) detecting the amplicon.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding specifically to the bentgrass eventASR-368 DNA in a sample are provided. Such methods comprising: (a)contacting the sample comprising DNA with a probe that hybridizes understringent hybridization conditions with genomic DNA from bentgrass eventASR-368 and does not hybridize under the stringent hybridizationconditions with a control bentgrass plant DNA; (b) subjecting the sampleand probe to stringent hybridization conditions; and (c) detectinghybridization of the probe to the ASR-368 DNA.

According to another aspect of the invention, methods of producing abentgrass plant that tolerates application of glyphosate are providedthat comprise the steps of: (a) sexually crossing a first parentalbentgrass event ASR-368 comprising the expression cassettes of thepresent invention, which confers tolerance to application of glyphosate,and a second parental bentgrass plant that lacks the glyphosatetolerance, thereby producing a plurality of progeny plants; and (b)selecting a progeny plant that tolerates application of glyphosate. Suchmethods may optionally comprise the further step of back-crossing theprogeny plant to the second parental bentgrass plant and selecting forglyphosate tolerant progeny to produce a true-breeding bentgrass varietythat tolerates application of glyphosate.

A turfgrass stand of grass that comprises bentgrass event ASR-368 isprovided. The turfgrass stand of bentgrass ASR-368 that is glyphosatetolerant is especially useful on a golf course and these turfgrassstands are an aspect of the invention.

Another aspect of the invention is a method for controlling weeds in aturfgrass stand of bentgrass ASR-368 comprising the step of applying aglyphosate containing herbicide formulation to the turfgrass stand.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Plasmid map of pMON25496

FIG. 2. Genomic organization of insert in Bentgrass event ASR-368

FIG. 3. ASR-368 5′ transgene/genomic DNA sequence (SEQ ID NO:3)

FIG. 4. ASR-368 3′ transgene/genomic DNA sequence (SEQ ID NO:4)

FIG. 5. ASR-368 5′ transgene/genomic junction region (SEQ ID NO:1) and3′ transgene/genomic junction region (SEQ ID NO:2)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin,Genes V, Oxford University Press: New York, 1994. The nomenclature forDNA bases as set forth at 37 CFR § 1.822 is used.

As used herein, the term “bentgrass” means Agrostis stolonifera andincludes all plant varieties that can be bred with bentgrass ASR-368.

As used herein, the term “comprising” means “including but not limitedto”.

“Glyphosate” refers to N-phosphonomethylglycine and its salts,Glyphosate is the active ingredient of Roundup® herbicide (MonsantoCo.). Treatments with “glyphosate herbicide” refer to treatments withthe Roundup®, Roundup Ultra®, Roundup Pro® herbicide or any otherherbicide formulation containing glyphosate. Examples of commercialformulations of glyphosate include, without restriction, those sold byMonsanto Company as ROUNDUP®, ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX,ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP® BIOFORCE,RODEO®, POLARIS®, SPARK® and ACCORD® herbicides, all of which containglyphosate as its isopropylammonium salt; those sold by Monsanto Companyas ROUNDUP® DRY and RIVAL® herbicides, which contain glyphosate as itsammonium salt; that sold by Monsanto Company as ROUNDUP® GEOFORCE, whichcontains glyphosate as its sodium salt; and that sold by Zeneca Limitedas TOUCHDOWN® herbicide, which contains glyphosate as itstrimethylsulfonium salt.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, i.e., a nucleic acid construct that includes atransgene of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another event that includethe heterologous DNA. Even after repeated back-crossing to a recurrentparent, the inserted DNA and flanking genomic DNA from the transformedparent is present in the progeny of the cross at the same chromosomallocation. The term “event” also refers to DNA from the originaltransformant comprising the inserted DNA and flanking genomic sequenceimmediately adjacent to the inserted DNA, that would be expected to betransferred to a progeny that receives the inserted DNA including thetransgene of interest as the result of a sexual cross of one parentalline that includes the inserted DNA (e.g., the original transformant andprogeny resulting from selling) and a parental line that does notcontain the inserted DNA. A glyphosate tolerant bentgrass plant can bebred by first sexually crossing a first parental bentgrass plantconsisting of a bentgrass plant grown from the transgenic bentgrassplant derived from transformation with the plant expression cassettescontained in pMON25496 (FIG. 1) that tolerates application of glyphosateherbicide, and a second parental bentgrass plant that lacks thetolerance to glyphosate herbicide, thereby producing a plurality offirst progeny plants; and then selecting a first progeny plant that istolerant to application of glyphosate herbicide; and selfing the firstprogeny plant, thereby producing a plurality of second progeny plants;and then selecting from the second progeny plants, a glyphosateherbicide tolerant plant. These steps can further include theback-crossing of the first glyphosate tolerant progeny plant or thesecond glyphosate tolerant progeny plant to the second parentalbentgrass plant or a third parental bentgrass plant, thereby producing abentgrass plant that tolerates the application of glyphosate herbicide.In the present invention, the transgenic bentgrass event is also definedas bentgrass event ASR-368 and may be referred to herein as ASR-368 orevent ASR-368.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, e.g., Fehr, inBreeding Methods for Cultivar Development, Wilcox J. ed., AmericanSociety of Agronomy, Madison Wis. (1987).

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid, in the case of thepresent invention, to a strand of genomic DNA from bentgrass eventASR-368 whether from a bentgrass event ASR-368 plant or from a samplethat includes DNA from the event. Probes according to the presentinvention include not only deoxyribonucleic or ribonucleic acids butalso polyamides and other probe materials that bind specifically to atarget DNA sequence and can be used to detect the presence of thattarget DNA sequence.

“Primers” are isolated nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. Primerpairs of the present invention refer to their use for amplification of atarget nucleic acid sequence, e.g., by the polymerase chain reaction(PCR) or other conventional nucleic acid amplification methods.

Probes and primers are generally 11 polynucleotides or more in length,often 18 polynucleotides or more, 24 polynucleotides or more, or 30polynucleotides or more. Such probes and primers are selected to be ofsufficient length to hybridize specifically to a target sequence underhigh stringency hybridization conditions. Preferably, probes and primersaccording to the present invention have complete sequence similaritywith the target sequence, although probes differing from the targetsequence that retain the ability to hybridize to target sequences may bedesigned by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR-primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as Primer(Version 0.5, © 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

Primers and probes based on the flanking genomic DNA and insertsequences disclosed herein can be used to confirm (and, if necessary, tocorrect) the disclosed DNA sequences by conventional methods, e.g., byre-cloning and sequencing such DNA molecules isolated from bentgrassASR-368 the seed of which is deposited with the ATCC having accessionnumber PTA-4816.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA molecule. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic event in a sample.Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure. Anucleic acid molecule is said to be the “complement” of another nucleicacid molecule if they exhibit complete complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to be“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989, and by Haymes et al.,In: Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C. (1985), Departures from complete complementarity aretherefore permissible, as long as such departures do not completelypreclude the capacity of the molecules to form a double-strandedstructure. In order for a nucleic acid molecule to serve as a primer orprobe it need only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In a preferredembodiment, a nucleic acid of the present invention will specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NO: 1, 2, 3, or 4, complements thereof or fragments of either undermoderately stringent conditions, for example at about 2.0×SSC and about65° C. In a particularly preferred embodiment, a nucleic acid of thepresent invention will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NO:1 through SEQ ID NO:4 orcomplements or fragments of either under high stringency conditions. Inone aspect of the present invention, a preferred marker nucleic acidmolecule of the present invention has the nucleic acid sequence setforth in SEQ ID NO:1 through SEQ ID NO:4 or complements thereof orfragments of either. In another aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 80% and 100% or 90% and 100% sequence identity with the nucleicacid sequence set forth in SEQ ID NO:1 through SEQ ID NO:4 or complementthereof or fragments of either. In a further aspect of the presentinvention, a preferred marker nucleic acid molecule of the presentinvention shares between 95% and 100% sequence identity with thesequence set forth in SEQ ID NO:1 through SEQ ID NO:4 or complementthereof or fragments of either. SEQ ID NO:1 through SEQ IN NO:4 may beused as markers in plant breeding methods to identify the progeny ofgenetic crosses similar to the methods described for simple sequencerepeat DNA marker analysis, in “DNA markers: Protocols, applications,and overviews: (1997) 173-185, Cregan, et al., eds., Wiley-Liss NY; allof which is herein incorporated by reference in its' entirely. Thehybridization of the probe to the target DNA molecule can be detected byany number of methods known to those skilled in the art, these caninclude, but are not limited to, fluorescent tags, radioactive tags,antibody based tags, and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofpolynucleic acid amplification of a target polynucleic acid moleculethat is part of a polynucleic acid template. For example, to determinewhether a bentgrass plant resulting from a sexual cross containstransgenic event genomic DNA from the bentgrass event ASR-368 plant ofthe present invention, DNA that is extracted from a bentgrass planttissue sample may be subjected to polynucleic acid amplification methodusing a primer pair that includes a primer derived from flanking DNA inthe genome of the ASR-368 plant adjacent to the insertion site of theinserted heterologous DNA (transgenic DNA), and a second primer derivedfrom the inserted heterologous DNA to produce an amplicon that isdiagnostic for the presence of the ASR-368 event DNA. The amplicon is ofa length and has a polynucleotide sequence that is also diagnostic forthe event. The amplicon may range in length from the combined length ofthe primer pairs plus one nucleotide base pair, preferably plus aboutfifty nucleotide base pairs, more preferably plus about twohundred-fifty nucleotide base pairs, and even more preferably plus aboutfour hundred-fifty nucleotide base pairs or more. Alternatively, aprimer pair can be derived from flanking genomic sequence on both sidesof the inserted heterologous DNA so as to produce an amplicon thatincludes the entire insert polynucleotide sequence (e.g., a forwardgenomic primer from SEQ ID NO:3 and a reverse genomic primer from SEQ IDNO:4 that amplifies an inserted DNA molecule comprising the HindIIIexpression cassette of pMON25496 DNA fragment that was transformed intobentgrass, about 6681 nucleotide base pairs, FIG. 1). A member of aprimer pair derived from the plant genomic sequence of ASR-368 may belocated a distance from the inserted DNA molecule, this distance canrange from one nucleotide base pair up to about twenty thousandnucleotide base pairs. The use of the term “amplicon” specificallyexcludes primer dimers that may be formed in the DNA thermalamplification reaction.

Polynucleic acid amplification can be accomplished by any of the variouspolynucleic acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A event of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods andApplications, ed. Innis et al., Academic Press, San Diego, 1990. PCRamplification methods have been developed to amplify up to 22 kb ofgenomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc.Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as othermethods known in the art of DNA amplification may be used in thepractice of the present invention. The sequence of the heterologous DNAinsert or flanking genomic DNA from bentgrass event ASR-368 can beverified (and corrected if necessary) by amplifying such DNA moleculesfrom the event using primers derived from the sequences provided hereinfollowed by standard DNA sequencing of the PCR amplicon or of the clonedDNA. DNA detection kits that are based on DNA amplification methodscontain DNA primers that specifically amplify a diagnostic amplicon. Thekit may provide an agarose gel based detection method or any number ofmethods of detecting the amplicon known in the art.

The amplicon produced by these methods may be detected by a plurality oftechniques. One such method is Genetic Bit Analysis (Nikiforov, et al.Nucleic Acid Res. 22:4167-4175, 1994), where a DNA oligonucleotide isdesigned that overlaps both the adjacent flanking genomic DNA sequenceand the inserted DNA sequence. The oligonucleotide is immobilized inwells of a microtiter plate. Following PCR of the region of interest(using one primer in the inserted sequence and one in the adjacentflanking genomic sequence), a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelleddideoxynucleotide triphosphate (ddNTPs) specific for the expected nextbase. Readout may be fluorescent or ELISA-based. A signal indicatespresence of the insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking genomic sequence) and incubated in the presenceof a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. Deoxyribonucleotides (DNTPs) are addedindividually and the incorporation results in a light signal that ismeasured. A light signal indicates the presence of the transgeneinsert/flanking sequence due to successful amplification, hybridization,and single or multi-base extension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon ofthe present invention. Using this method an oligonucleotide is designedthat overlaps the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence) and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. Single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene insert/flanking sequence due tosuccessful amplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps thegenomic flanking and insert DNA junction. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996) Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization.

Bentgrass event ASR-368 is tolerant to glyphosate herbicide and isuseful as a turfgrass stand. A turfgrass stand is cultivated in privateand public areas. A good turfgrass stand, or green, has both beauty andusefulness; its maintenance for golf, tennis, baseball, football, andother sports fields is a costly and specialized procedure. The bentgrassASR-368 event is especially useful as a turfgrass stand grown on golfcourses. Golf courses have various turfgrass stand turfgrass componentsthat make up a hole. These components include the tee, the fairway, therough and the green. Event ASR-368 when used as a turfgrass provides aturfgrass stand that can be effectively managed for weed control by theapplication of a glyphosate containing herbicide. A turfgrass standcomprising the bentgrass event ASR-368 is an aspect of the invention,whereas the ASR-368 turfgrass stand is a component of a golf course,then that component is an aspect of the invention. A turfgrass stand ofthe present invention preferably comprises bentgrass event ASR-368 as a50 percent or more component, more preferably a 75 percent component,and even more preferably greater than a 90 percent component.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1

The transgenic bentgrass event ASR-368 was generated by microprojectilebombardment of bentgrass line B99061R/990028 using a linear HindIII DNAfragment derived from pMON25496 (FIG. 1) that comprises the transgeneinsert of the present invention. This DNA fragment contains twotransgene expression cassettes that collectively confer bentgrassASR-368 plant tolerance to glyphosate. The first cassette is composed ofthe rice actin 1 promoter and intron (P-Os.Act1, also referred to asP-ract, and the intron I-Os.Act1, also referred to as ract intron, U.S.Pat. No. 5,641,876), operably connected to an Arabidopsis EPSPSchloroplast transit peptide (TS-At.EPSPS:CTP2, also referred to as ctp2,Klee et al., Mol. Gen. Genet. 210:47-442, 1987), operably connected to aglyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS)from Agrobacterium sp. strain CP4 (AGRTU.aroA:CP4 EPSPS, also known ascp4, U.S. Pat. No. 5,633,435) and operably connected to a nopalinesynthase transcriptional terminator (T-nos, also referred to as NOS 3′,Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803-4807, 1983). The secondtransgene expression cassette consists of the cauliflower mosaic virus35S promoter containing a tandem duplication of the enhancer region(P-CaMV.35S, also referred to as P-e35S, Kay et al. Science236:1299-1302, 1987; U.S. Pat. No. 5,164,316), operably connected to aZea mays Hsp70 intron (I-Zm.Hsp70, also referred to as ZmHSP70 intron,U.S. Pat. No. 5,362,865), operably connected to an Arabidopsis EPSPSchloroplast transit peptide (TS-At.EPSPS:CTP2), operably connected to aglyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS)from Agrobacterium sp. strain CP4 (AGRTU.aroA:CP4 EPSPS) and operablyconnected to a nopaline synthase transcriptional terminator (T-nos,Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803-4807, 1983). The DNAconstruct pMON25496 has been shown to confer glyphosate tolerance intransgenic corn (US 20020013960 A1). Post-bombardment,glyphosate-tolerant transgenic calli were selected on media containing 3mM glyphosate and plants were subsequently regenerated. Transgenicevents were produced and event ASR-368 was selected from this populationbased on a superior combination of characteristics, including glyphosatetolerance, agronomic performance, and single transgenic insertion. Thetransgene insertion as it occurs in ASR-368 is shown in FIG. 2.

Example 2

The glyphosate tolerant bentgrass event ASR-368 was tested for toleranceto glyphosate vegetative injury. The glyphosate tolerant bentgrass eventASR-368 showed no damage to 5% Roundup® Pro (glyphosate containingherbicide formulation) sprayed with a hand sprayer or an amountequivalent to 128 ounces Roundup® Pro per acre. The standard recommendedrate is 1.25 to 2.5% Roundup® Pro or amount equivalent to 32 to 64ounces Roundup® Pro per acre. Three applications of the glyphosatecontaining herbicide formulation during the growing season, earlysummary, mid-summer and early fall were used to test for glyphosatetolerance in a turfgrass stand of event ASR-368. Bentgrass event ASR-368showed glyphosate tolerance to all applications of glyphosate at threetest locations. No vegetative injury was observed on event ASR-368,while bentgrass plants not containing pMON25496 were all heavily injuredor killed by the glyphosate containing herbicide formulation treatment.Treatment of bentgrass ASR-368 that is a turfgrass component of aturfgrass stand with a glyphosate containing herbicide is a methoduseful for controlling weeds and other unwanted plants in the turfgrassstand.

Example 3

The DNA sequences of the 5′ and 3′ genomic regions adjacent to thetransgene insert are determined by isolation of the DNA molecules usingClontech's Universal Genome Walker™ Kit and the RAGE method (RapidAmplification of Genomic DNA Ends). The 5′ transgene/genomic DNA (FIG.3) is isolated from bentgrass ASR-368 genomic DNA by: digestionovernight at 37° C. with HindIII, and digestion of pBluescript KSplasmid (Stratagene, La Jolla Calif.) for 3 hours at 37° C. with XbaI.The four nucleotide base overhangs are filled in with two nucleotides tobecome compatible for ligation. The genomic DNA is ligated with the XbaIdigested/2 nucleotide base filled pBluescript KS plasmid by incubationwith T4 DNA ligase under the appropriate conditions. After ligationreaction, 5 μl of the ligation mix is used in a DNA amplification methodwith 2 μl of 10 μM M13 forward primer (SEQ ID NO:5), 2 μl 10 μM ASR-368transgene-specific oligonucleotide primer (SEQ ID NO:6), 1.75 μl 10 mMdeoxyribonucleotides, the Expand Long Template PCR System (Roche) andwater to 50 μl. A primary reaction is performed in a thermocycler withthe following cycling conditions: 30 cycles of 94 C for 2 minutes; 94 Cfor 10 seconds each; 56 C for 30 seconds, 68 C for 3 minutes; andfinally 68 C for 10 minutes. One μl of the primary reaction is amplifiedin a secondary reaction that includes 2 μl of 10 μM T7 primer (SEQ IDNO:7), 2 μl of ASR-368 specific primer (SEQ ID NO:8), 1.75 μl 10 mMdeoxyribonucleotides, the Expand Long Template PCR System (Roche) andwater to 50 μl, the thermocycler conditions are the same as used for theprimary reaction.

The presence of the transgene/genomic DNA in a bentgrass sample isverified by PCR. The 5′ transgene/genomic junction region amplicon isproduced using one primer (SEQ ID NO:11), designed to the genomic DNAsequence flanking the 5′ end of the insert paired with a second primer(SEQ ID NO:12) in the rice actin 1 promoter of the inserted transgeneDNA. The 5′ junction amplicon is produced from about 50 ng of leafgenomic DNA (1 μl) as a template, 15 pmol of each primer (1.5 μl each),and the Expand High Fidelity PCR system in a 50 μl reaction volume. Theamplification of the reactions was performed under the following cyclingconditions: 1 cycle at 94° C. for 2 minutes; 10 cycles at 94° C. for 15seconds, 60° C. for 30 seconds, 72° C. for 1 minute; 25 cycles at 94° C.for 15 seconds, 60° C. for 30 seconds 72° C. for 1 minute+5 additionalseconds per cycle; 1 cycle 72° C. for 7 minutes.

In another method, the isolation of the corresponding transgene/genomicDNA molecules from bentgrass event ASR-368 can also be accomplishedusing ligated adapters and nested PCR as described in the Genome Walker™kit (catalog #K1807-1, CloneTech Laboratories, Inc, Palo Alto, Calif.).First, genomic DNA from the ASR-368 event is isolated by the CTABpurification method (Rogers et al., Plant Mol. Biol. 5:69-76, 1985). Thegenomic DNA libraries for amplification are prepared according tomanufacturer's instructions (Genome Walker™, CloneTech Laboratories,Inc, Palo Alto, Calif.). In separate reactions, genomic DNA is digestedovernight at 37° C. with blunt-end restriction endonucleases (CloneTechLaboratories, Inc, Palo Alto, Calif.). The reaction mixtures areextracted with phenol:chloroform, the DNA is precipitated by theaddition of ethanol to the aqueous phase, pelleted by centrifugation,then resuspended in Tris-EDTA buffer (10 mM Tris-.HCl, pH 8.0, 1 mMEDTA). The purified blunt-ended genomic DNA fragments are ligated to theGenome Walker™ adapters according to the manufacturer's protocol. Afterligation, each reaction is heat treated (70° C. for 5 min) to terminatethe reaction and then diluted 10-fold in Tris-EDTA buffer. One μl ofeach respective ligation is then amplified in a 50 μl reaction thatincluded 1 μl of respective adapter-ligated library, 1 μl of 10 μMGenome Walker™ adapter primer AP1 (SEQ ID NO:9, supplied bymanufacturer), 1 μl of 10 μM event ASR-368 transgene-specificoligonucleotide (SEQ ID NO:12), 1 μl 10 mM deoxyribonucleotides, 2.5 μldimethyl sulfoxide, 5 μl of 10× PCR buffer containing MgCl₂, 0.5 μl (2.5units)of Amplitaq thermostable DNA polymerase (PE Applied Biosystems,Foster City, Calif.), and H₂O to 50 μl. The reactions are performed in athermocycler using calculated temperature control and the followingcycling conditions: 1 cycle of 95° C. for 9 minutes; 7 cycles of 94° C.for 2 seconds, 70° C. for 3 minutes; 36 cycles of 94° C. for 2 seconds,65° C. for 3 minutes; 1 cycle of 65° C. for 4 minutes. One μl of eachprimary reaction is diluted 50-fold with water and amplified in asecondary reaction (1 μl of respective diluted primary reaction, 1 μl of10 μM Genome Walker™ nested adapter primer AP2, (SEQ ID NO: 10, suppliedby manufacturer), 1 μl of 10 μM event ASR-368 transgene-specific nestedoligonucleotide (SEQ ID NO:12), 1 μl 10 mM deoxyribonucleotides, 2.5 μldimethyl sulfoxide, 5 μl of 10×PCR buffer containing MgCl₂, 0.5 μl (2.5units) of Amplitaq thermostable DNA polymerase (PE Applied Biosystems,Foster City, Calif.), and H₂O to 50 μl) using the following cyclingconditions: 1 cycle of 95° C. for 9 minutes; 5 cycles of 94° C. for 2seconds, 70° C. for 3 minutes; 24 cycles of 94° C. for 2 seconds, 65° C.for 3 minutes; 1 cycle of 65° C. for 4 minutes.

PCR products, representing 5′ regions that span the junction between thebentgrass event ASR-368 transgenic insertion and the neighboringflanking bentgrass genomic DNA sequence are purified by agarose gelelectrophoresis followed by isolation from the agarose matrix using theQIAquick Gel Extraction Kit (catalog #28704, Qiagen Inc., Valencia,Calif.) and direct cloning into the pGEM-T Easy vector (catalog. #A1360,Promega, Madison, +Wis.). The identity of the cloned PCR products andrelationship to the HindIII fragment of pMON25496 that was used toproduce bentgrass ASR-368 is confirmed by DNA sequence analysis (ABIPrism™ 377, PE Biosystems, Foster City, Calif. and DNASTAR sequenceanalysis software, DNASTAR Inc., Madison, Wis.). The DNA sequence of the5′ genomic/transgene region DNA molecule is illustrated in FIG. 3. FIG.3 further identifies the bentgrass genomic DNA portion by showing it asunderlined DNA sequence, the double underlined DNA sequence is DNAsequence homologous or complementary to PCR primer molecules useful inthe identification of a bentgrass genome that contains SEQ ID NO:3.

Similarly, the bentgrass event ASR-368 3′ flanking genomic DNA sequence(FIG. 4) is amplified using one primer (SEQ ID NO:14) designed to thegenomic DNA sequence flanking the 3′ end of the transgene insert and asecond primer (SEQ ID NO:13) located in the T-nos 3′ transcriptiontermination region contained in pMON25496. The PCR is conducted usingabout 211 ng of leaf genomic DNA (1 μl) as a template, 15 pmol of eachprimer (1.5 μl each), and the Expand Long Template PCR system (Roche) ina 50 μl reaction volume. The amplification of the reactions is performedunder the following cycling conditions: 1 cycle at 94° C. for 2 minutes;35 cycles at 94° C. for 10 seconds, 60° C. for 30 seconds, 68° C. for 30seconds; 1 cycle at 68° C. for 10 minutes.

Bentgrass genomic DNA sequence flanking both sides of the transgenicinsertion was determined for event ASR-368 by sequencing the GenomeWalker™-derived amplification products and alignment to known transgenesequence. A 5′ region of the transgene insertion site was sequenced,this region comprises a transgene/genomic DNA sequence of 896 nucleotidebase pairs (bps) (SEQ ID NO:3) around the insertion junction. This DNAsequence consists of 637 bps of the flanking bentgrass genomic sequence(nucleotides 1-637 of SEQ ID NO:3), and 259 bps of sequence from the 5′end of P-Os.Actl (nucleotides 638-896 of SEQ ID NO:3) as shown in FIG.3.

The DNA sequence was determined for a 474 bps segment (SEQ ID NO:4)around the 3′ insertion junction, which from the 5′ end of the segmenthas 248 bps of the T-nos transcriptional terminator (nucleotides 1-248of SEQ ID NO:4), and the remaining sequence consisting of bentgrassgenomic DNA sequence flanking the integration site (corresponding tobases 249-474 of SEQ ID NO:4) as shown in FIG. 4. The double underlinedDNA sequence is DNA sequence homologous or complementary to PCR primermolecules useful in the identification of a bentgrass genome thatcontains SEQ ID NO:4

The junction sequences, SEQ ID NO:1 and SEQ ID NO:2 (FIG. 5) are novelDNA sequences from event ASR-368 and are diagnostic for bentgrass plantevent ASR-368 and its progeny. The junction sequences in SEQ ID NO:1 andSEQ ID NO:2 comprise polynucleotides on each side of an insertion siteof a transgene sequence fragment and bentgrass genomic DNA. The junctionsequence SEQ ID NO:1 is found at nucleotide position 626-649 of SEQ IDNO:3, the 5′ region of the transgene insertion site. The junctionsequence SEQ ID NO:2 is located at nucleotide position 236-259 of SEQ IDNO:4, the 3′ region of the transgene insertion site. Either junctionsequence can be used as a DNA probe or primer to specifically identifygenomic DNA of event ASR-368.

Example 4

DNA event primer pairs are used to produce an amplicon diagnostic forbentgrass event ASR-368. Amplicons diagnostic for ASR-368 comprise atleast one junction sequence, SEQ ID NO:1 or SEQ ID NO:2. ASR-368 eventprimer pairs that will produce a diagnostic amplicon for bentgrassASR-368 include, but are not limited to a primer pair that includesevent primer 1 (SEQ ID NO:11) and event primer 2 (SEQ ID NO:12) thatprovide a 5′ amplicon DNA molecule, and a primer pair, SEQ ID NO:13 andSEQ ID NO:14 that when substituted for primers 1 and 2 in the protocoloutlined in Table 1 produce the 3′ amplicon DNA molecule. In addition tothese primer pairs, any primer pair derived from SEQ ID NO:3 or SEQ IDNO:4 that in a DNA amplification reaction produces an amplicondiagnostic for bentgrass event ASR-368 is an aspect of the presentinvention. Any single isolated DNA polynucleotide primer moleculecomprising at least 11 contiguous nucleotides of SEQ ID NO:3, or itscomplement that is useful in a DNA amplification method to produce anamplicon diagnostic for bentgrass event ASR-368 is an aspect of theinvention. Any single isolated DNA polynucleotide primer moleculecomprising at least 11 contiguous nucleotides of SEQ ID NO:4, or itscomplement that is useful in a DNA amplification method to produce anamplicon diagnostic for bentgrass event ASR-368 is an aspect of theinvention. The amplification conditions for this analysis areillustrated in Table 1 and Table 2, however, any modification of thesemethods that use DNA primers to produce an amplicon diagnostic forbentgrass event ASR-368 is within the ordinary skill of the art. Adiagnostic amplicon comprises at least one transgene/genomic junctionDNA (SEQ ID NO:1 or SEQ ID NO:2).

An analysis for event ASR-368 plant tissue sample should include apositive tissue control from event ASR-368, a negative control from abentgrass plant that is not event ASR-368, and a negative control thatcontains no bentgrass DNA. Additional primer sequences can be selectedfrom SEQ ID NO:3 and SEQ ID NO:4 by those skilled in the art of DNAamplification methods, and conditions selected for the production of anamplicon may the methods shown in Table 1 and Table 2 or differ, butresult in an amplicon diagnostic for event ASR-368. The use of these DNAprimer sequences with modifications to the methods of Table 1 and 2 arewithin the scope of the invention. The amplicon produced by at least oneDNA primer sequence derived from SEQ ID NO:3 or SEQ ID NO:4 that isdiagnostic for ASR-368 is an aspect of the invention.

DNA detection kits that contain at least one DNA primer derived from SEQID NO:3 or SEQ ID NO:4 that when used in a DNA amplification methodproduces a diagnostic amplicon for bentgrass ASR-368 is an aspect of theinvention. The amplicon produced by at least one primer sequence derivedfrom any of the genetic elements of pMON25496 that is diagnostic forASR-368 is an aspect of the invention. A bentgrass plant or seed,wherein its genome will produce an amplicon diagnostic for bentgrassevent ASR-368 when tested in a DNA amplification method to amplify a DNAmolecule from DNA extracted from said bentgrass plant or seed is anaspect of the invention. The assay for the ASR-368 amplicon can beperformed by using a Stratagene Robocycler, MJ Engine, Perkin-Elmer9700, or Eppendorf Mastercycler Gradient thermocycler as shown in Table2, or by methods and apparatus known to those skilled in the art.

TABLE 1 PCR procedure and reaction mixture conditions for theidentification of bentgrass event ASR-368 5' transgene insert/genomicjunction region. Step Reagent Amount Comments 1 Nuclease-free water addto final — volume of 20 μl 2 10X reaction buffer 2.0 μl 1X final (withMgCl₂) concentration of buffer, 1.5 mM final concentration of MgCl₂ 3 10mM solution of 0.4 μl 200 μM final dATP, dCTP, dGTP, and concentrationof dTTP each dNTP 4 event primer 1 (SEQ ID 0.4 μl 0.2 μM final NO:11)(resuspended in concentration 1X TE buffer or nuclease-free water to aconcentration of 10 μM) 5 event primer 2 (SEQ ID 0.4 μl 0.2 μM finalNO:12) (resuspended in concentration 1X TE buffer or nuclease-free waterto a concentration of 10 μM) 6 RNase, DNase free 0.1 μl 50 ng/reaction(500 ng/μl) 7 REDTaq DNA poly- 1.0 μl 1 unit/reaction merase (1 unit/μl)(recommended to switch pipets prior to next step) 8 Extracted DNA —(template): Samples to be analyzed individual leaves 10-200 ng ofgenomic DNA pooled leaves (maximum 200 ng of of 50 leaves/pool) genomicDNA Negative control 50 ng of bentgrass genomic DNA (not ASR-368)Negative control no template DNA Positive control 50 ng of ASR-368genomic DNA

TABLE 2 Suggested PCR parameters for different thermocyclers Gently mixand, if needed (no hot top on thermocycler), add 1-2 drops of mineraloil on top of each reaction. Proceed with the PCR in a StratageneRobocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf MastercyclerGradient thermocycler using the following cycling parameters. Cycle No.Settings: Stratagene Robocycler 1 94° C. 3 minutes 38 94° C. 1 minute60° C. 1 minute 72° C. 1 minute and 30 seconds 1 72° C. 10 minutes CycleNo. Settings: MJ Engine or Perkin-Elmer 9700 1 94° C. 3 minutes 38 94°C. 10 seconds 60° C. 30 seconds 72° C. 1 minute 1 72° C. 10 minutesNote: The MJ Engine or Eppendorf Mastercycler Gradient thermocyclershould be run in the calculated mode. Run the Perkin-Elmer 9700thermocycler with the ramp speed set at maximum.

A deposit of the Monsanto Company, bentgrass seed ASR-368 disclosedabove and recited in the claims gas been made under the Budapest Treatywith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110. The ATCC accession number is PTA-4816.The deposit will be maintained in the depository for a period of 30years, or 5 years after the last request, or for the effective life ofthe patent, whichever is longer, and will be replaced as necessaryduring that period.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. A seed of bentgrass plant designated ASR-368, having representativeseed of said bentgrass plant having been deposited under ATCC AccessionNo. PTA-4816.
 2. A bentgrass plant ASR-368 or part thereof produced bygrowing the seed of claim
 1. 3. The bentgrass plant ASR-368 or partthereof of claim 2, comprising pollen, ovule, seed, roots, or leaves. 4.A progeny seed of the bentgrass plant ASR-368 of claim 2 wherein saidprogeny seed comprises a DNA molecule selected from the group consistingof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
 5. Abentgrass plant grown from said progeny seed of claim 4, wherein thegenome of said bentgrass plant comprises a DNA molecule selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQID NO:4.
 6. A method of producing a plant that tolerates application ofglyphosate herbicide comprising: (a) sexually crossing a firstglyphosate tolerant bentgrass plant ASR-368 having representative seedof said bentgrass plant deposited under ATCC Accession No. PTA-4816 anda second parent bentgrass plant that lacks the tolerance to glyphosateherbicide, thereby producing a plurality of progeny plants; and (b)selecting a progeny plant that is tolerant to application of glyphosate.7. The method of claim 6 further comprising the step of backcrossing thefirst progeny plant that is glyphosate tolerant or the second progenyplant that is glyphosate tolerant to the second parent plant or a thirdparent plant, thereby producing a plant that tolerates the applicationof glyphosate.
 8. A bentgrass plant, seed, or DNA-containing partthereof comprising bentgrass event ASR-368.
 9. A bentgrass plant, seed,or DNA-containing part thereof capable of producing an ASR-368diagnostic amplicon.
 10. The bentgrass plant, seed, or DNA-containingpart thereof of claim 9, wherein the ASR-368 diagnostic ampliconcomprises SEQ ID NO:1 or SEQ ID NO:2.